The present invention relates to polynucleotides which may be useful in recombinant plant DNA technology or analysis, in particular to tissue- or ripening-specific promoter DNA, and products and methods employing such DNA.
It is desirable to be able to specifically express (or inhibit the expression of) genes in plants, for instance in particular tissues, or at a particular developmental stage. This may allow particular biosynthetic enzymes to be produced only in the fruit of a plant, and not in other tissues wherein it may have undesirable effects. Likewise it may be desirable to have particular protective proteins (e.g. anti-fungal, pesticidal) expressed only during a particular vulnerable developmental stage e.g. early or late ripening.
This type of specific expression can be achieved by using inducible promoters which are xe2x80x98switched onxe2x80x99 in the presence of environmental signals present only in restricted tissues of the plant, or only at particular times. Such promoters have already been made available for tomatoes. Thus WO93/07257 (SPI Inc.) relates, inter alia, to gene-fusions capable of conferring tissue-specific or developmentally regulated gene constructs. These constructs apparently allow particular genes to be expressed during the formation and ripening of fruit. The coding region of clone xcexUC82-3.3 in WO93/07257, which was derived from tomato, has homology to a bacterial histidine decarboxylase (HDC). Similarly WO94/13797 (CSIRO) relates, inter alia, to inducible soft-fruit promoter DNA derived from alcohol dehydrogenase (ADH) in tomatoes. ADH apparently has a role in ripening in that it metabolises alcohols and aldehydes involved in flavour. The ADH promoter is apparently sensitive to and therefore inducible by high levels of O2.
It is clear from the foregoing that the disclosure of novel inducible promoters, particularly those active in plants other than tomato plants, would provide a useful contribution to the art.
The applicants have now isolated inducible promoters from apple, elements of which show useful properties and which may be useful in particular in the isolation of other ripening specific promoters or transcription factors, or in the genome mapping studies.
In a first aspect of the present invention there is disclosed a recombinant polynucleotide comprising a promoter sequence being: (a) an inducible promoter obtainable from apple, or (b) a functional portion therof, or (c) a functional derivative or homolog promoter being at least 70% homologous to either.
As used herein, xe2x80x9cpromoterxe2x80x9d refers to a non-coding region of DNA involved in binding of RNA polymerase and other factors that initiate or modulate transcription whereby an RNA transcript is produced. Promoters, depending upon the nature of the regulation, may be constitutive or inducible. A constitutive promoter is always turned on. An inducible promoter requires specific signals in order for it to be turned on or off. These may be particular signals for example chemical signals, which are applied to a cell under certain conditions or as a result of a deliberate application. In the context of the present application, the term xe2x80x9cinduciblexe2x80x9d is intended to include particularly promoters which are tissue-specific in that they are effective only in certain plant tissues either with or without externally applied inducing agents, or ripening specific promoters which switched on within some or all plant cells as a result of ripening, for example in response to ethylene produced during the ripening process.
Examples of promoters of the invention include a ABG1 xcex2-galactosidase promoter whose sequence is included within the sequence shown in FIG. 3 hereinafter (SEQ ID NO 1); and the ACC synthase promoter whose sequence is comprised within the sequence shown in FIG. 5 (SEQ ID NO 2) hereinafter.
Thus, the invention provides a promoter comprising at least a functional portion of the Sequence shown in FIG. 3 or FIG. 5.
As well as authentic promoters obtainable from apple, the invention also embraces functional portions thereof.
The term xe2x80x9cfunctionalxe2x80x9d is used herein to describe moieties which have the activity of a promoter as defined above, when present in apple cells.
Also embraced by present invention are functional derivative promoters being at least 70% homologous to the above.
By xe2x80x9cderivativexe2x80x9d is meant a sequence may be obtained by introducing changes into the full-length or part length sequence, for example substitutions, insertions, and/or deletions. This may be achieved by any appropriate technique, including restriction of the sequence with an endonuclease followed by the insertion of a selected base sequence (using linkers if required) and ligation. Also possible is PCR-mediated mutagenesis using mutant primers. Such changes may be introduced e.g. to remove or incorporate restriction sites into the sequence.
Also embraced by the present invention are functional xe2x80x9chomologsxe2x80x9d of authentic promoters obtained from apple which hybridise thereto and are at least 70% homologous to either the full-length or part length sequences and in particular to SEQ ID NOS 1 and 2 identified herein.
Such homologs may conveniently be identified and isolated by those skilled in the art from a test sample as follows:
The test sample is contacted with the apple promoter under suitable hybridisation conditions, and any test DNA (e.g. an apple genomic library) which hybridises thereto is identified.
Such screening is initially carried out under low-stringency conditions, which comprise a temperature of about 37xc2x0 C. or less, a formamide concentration of less than about 50%; and a moderate to low salt (e.g. Standard Saline Citrate (xe2x80x98SSCxe2x80x99)=0.15 M sodium chloride; 0.15 M sodium citrate; pH 7) concentration. Alternatively, a temperature of about 50xc2x0 C. or less and a high salt (e.g. xe2x80x98SSPExe2x80x99=0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferably the screening is carried out at about 37xc2x0 C., a formamide concentration of about 20%, and a salt concentration of about 5xc3x97SSC, or a temperature of about 50xc2x0 C. and a salt concentration of about 2xc3x97SSPE. These conditions will allow the identification of sequences which have a substantial degree of similarity with the probe sequence, without requiring perfect homology for the identification of a stable hybrid. The phrase xe2x80x98substantial similarityxe2x80x99 refers to sequences which share at least 50% overall sequence identity. Preferably, hybridisation conditions will be selected which allow the identification of sequences having at least 70k sequence identity with the probe, while discriminating against sequences which have a lower level of sequence identity with respect to the probe.
After low stringency hybridization has been used to identify one or more homologs having a substantial degree of similarity with the probe sequence, this subset is then subjected to high stringency hybridization, so as to identify those clones having a particularly high level of homology with respect to the probe sequences. High stringency conditions comprise a temperature of about 42C or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration. Alternatively they may comprise a temperature of about 65C or less, and a low salt (SSPE) concentration. Preferred conditions for such screening comprise a temperature of about 42C, a formamide concentration of about 20%, and a salt concentration of about 2xc3x97SSC, or a temperature of about 65C, and a salt concentration of about 0.2 SSPE.
Thus, according to the present invention the derivative sequence or homolog is at least 70% identical to the sequence of the full or part-length promoters. Typically there is 80% or more, 90% or more 95% or more or 98% or more identity between the derivative or homolog and the authentic sequences. There may be up to five, for example up to ten or up to twenty nucleotide deletions, insertions and/or substitutions made to the full-length or part length sequences.
Whether a part-length or modified or homologous sequence is capable of acting as a promoter (is xe2x80x9cfunctionalxe2x80x9d) may be readily ascertained in the light of the present disclosure by those skilled in the art. Briefly, the candidate sequence is provided in a vector upstream of a protein coding sequence at a position in which it is believed to be operatively linked to that coding sequence. A suitable host cell, preferably an apple cell, is transformed with the resulting vector. The presence or absence of the protein coded by the sequence is determined.
Preferably the polynucleotide of the first aspect comprises a promoter sequence which is activated in response to tissue specific agents i.e. is turned on or off as a function of the tissue in which it is present. More preferably the agents are specific to fruit, and most preferably specific to ripening is fruit (i.e. the promoter is a developmentally regulated promoter which is turned on or off as a function of development).
Two particular examples of promoter sequences of the invention are the Apple xcex2-Galactosidase (ABG1) promoter, or the 1-AminoCyclopropane-1-Carboxylate synthase (ACC Synthase) promoter. Isolated, non-recombinant, polynucleotides encoding these promoters, or functional portions or dervatives or homologs thereof form a further part of the present invention. The sequences of these promoters are included within the sequences given hereinafter in FIGS. 3 and 5 respectively and recombinantly produced or synthetic promoters comprising or derived from these sequences also fall within the ambit of the invention.
Computer-assisted examination of the DNA sequences of the ABG1 (2879-bp) and the AAS ACC synthase (5391-bp) promoter containing fragments of FIGS. 3 and 5 has shown the presence of some interesting sequence motifs as illustrated in FIGS. 7 and 8 below (SEQ ID NOS 3 and 4 and 5 and 6 respectively). These motifs form preferred examples of portions of the ABG1 and AAS ACC synthase promoters.
A: At approximately the same location (1.5-1.6-kbp) upstream from the start codon of these two ripening-related genes there is a highly conserved sequence of 155-bp. The orientation of the sequence is opposite in the two promoters (SEQ ID NOS 3 and 4). These two sequences are 90% similar and contain an unusual repeat element (GAAAAATCACATTTTTACACTAAAAAG-SEQ ID NO 7) or a derivative thereof, which has dyad symmetry about the central T residue. This unit is found in the ACC synthase promoter sequence (FIG. 8) and is varied only by two conservative (Txe2x86x92C) substitutions in the ABG1 sequence. This is believed to be the binding site for a dimeric transcription factor, and considering the extent of conservation of the DNA sequence encompassing this motif, it may be involved in the regulation of transcription during fruit ripening.
This 155 bp DNA sequence could be used as a probe fragment to isolate other ripening-specific promoters by library screening, for example as described above.
Furthermore, as it is likely to be important in ripening-specific gene expression, the sequence could be used as a component of a minimal promoter. Removal of extraneous non-functional sequences is desirable to satisfy regulatory considerations and would reduce the size of promoters considerably, making them more versatile.
Thus in a preferred embodiment, the invention provides a inducible promoter which comprises SEQ ID NO 3 or SEQ ID NO 4 or a functional portion thereof, or a functional derivative or homolog promoter being at least 70!k homologous to either. Preferably, the promoter will comprise SEQ ID NO 3 of SEQ ID NO 4.
These sequences could be used in strategies to isolate transcription factors involved in ripening-specific gene expression. They could be coupled to magnetic beads to affinity purify proteinaceaous factors from extracts of fruit cell nuclei or could be radiolabelled and used to screen a fruit cDNA expression library. Such methods form a further aspect of the invention.
B: Another notable sequence occurs approximately 4.7-kbp upstream of the start codon in the ACC synthase promoter (FIG. 7). This sequence (SEQ ID NO 5 in FIG. 8) of 227-bp has Inverted repeat (IR) elements at its termini. The only significant similarity identified is with a sequence (217-bp) seen in the promoter of an apple kn1-like knotted gene homologue [Watillon, B. (1996), M.domestica partial gene for kn1-like protein. GB accession Z71981- SEQ ID NO 6]. The homology is 61% overall, but considerably higher at the termini.
A PCR fragment encompassing the apple kn1-like IR element has been used to probe a Southern blot of genomic DNA. This showed that there are multiple copies of the element in the apple genome and appears to confirm that the sequences represent transposable inverted repeat elements. The identification of such elements has never before been reported in apple.
These elements share some features with the Stowaway class of IR elements [Bureau, T. E. and Wessler, S. R. (1994) Stowaway: A new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants. The Plant Cell 6: 907-916]. Stowaway and similar plant IR elements may represent transposable elements, their remnants after transposition or solo terminal repeats from a larger element.
The apple IR element identified is similar in size to Stowaway elements found in dicotyledonous plants (248 bp+/xe2x88x9224 bp) and is also AT rich. It differs in the target site for insertion (TA in Stowaway) and the nature of the conserved terminal repeat region. It therefore represents a new class of element which has not been reported previously.
The inverted repeat element may be of use in genome mapping in rosaceous species. Depending on how widespread it is and in what copy numbers it is found, it may be used in a similar way to microsatellites. Such methods form yet a further aspect of the invention.
In a further aspect of the invention there is provided a replication vector comprising a polynucleotide as described above and further comprising a replication element which permits replication of the vector in a suitable host cell.
xe2x80x9cVectorxe2x80x9d is defined to include, inter alia, any plasmid DNA, lysogenic phage DNA and/or transposon DNA, in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication). Introduced by any method e.g. conjugation, mobilisation, transformation, transfection, transduction or electroporation. The term explicitly includes shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in both bacterial and plant cells.
In yet a further aspect of the invention there is provided an expression vector comprising a polynucleotide as described above. Preferably the vector further comprises a heterologous gene operatively linked to said promoter sequence.
As used herein, the terms xe2x80x9coperatively linkedxe2x80x9d denotes the linkage of a promoter or a non-coding gene regulatory sequence to an RNA-encoding DNA sequence, and especially to the ability of the regulatory sequence or promoter to induce production of RNA transcripts corresponding to the DNA-encoding sequence when the promoter or regulatory sequence is recognised by a suitable polymerase.
Preferably the heterologous gene encodes any of: (a) antisense RNA capable of down-regulating genes involved in ripening; (b) a peptide or protein improving fungal, insect, bacterial, viral, herbicidal, nematode, or arachnid resistance; (c) a detectable or selectable marker protein. Examples of some of such heterologous genes are known to those skilled in the art (see e.g. WO93/07257, WO94/13797). Ripening specific genes include those involved in ethylene biosynthesis or cell wall degradation. Proteins involved in fungal degradation include xcex2-1,3-glucanases and chitinases. Marker proteins include xcex2-glucuronidase (GUS).
Preferably the vector comprises elements derived from disarmed strains of Agrobacterium tumefaciens, such as are known to those in the art.
The invention further provides a host cell containing a vector as described claimed above, or transformed with such a vector. Typically the host cell will constitute all or part of a plant protoplast, plant callus, plant tissue, developing plantlet, or immature whole plant. The plants/cells may be apple or other fruit in which the promoters are functional (e.g. tomato, melon, strawberry).
In addition, the invention provides a method of producing a transgenic plant comprising regenerating a mature plant from the transformed host cell described above.
As used herein, xe2x80x9ctransgenicxe2x80x9d plants refer to plants or plant is compositions in which heterologous or foreign DNA is expressed or in which the expression of a gene naturally present in the plant has been altered. Such heterologous DNA will be in operative linkage with plant regulatory signals and sequences. The DNA may be integrated into a chromosome or integrated into an episomal element, such as the chloroplast, or may remain as an episomal element. In creating transgenic plants or plant compositions, any method for introduction of such DNA known to those of skill in the art may be employed. A transgenic plant comprising such a host cell, either produced as described above or by further propagation of transgenic plants forms a sixth aspect of the invention.
A further aspect of the invention provides a method of producing apples having a modified phenotype, said method comprising cultivating a transgenic apple plant described above and harvesting the fruit of the plant. The fruit itself forms yet a further aspect of the invention.
The invention will now be further described with reference to the following non-limiting examples. Further embodiments falling within the scope of the invention will occur to those skilled in the art in the light of these.